1. Field of the Invention
This invention relates to a process for producing and recovering polypeptides from bacterial cells. More particularly, this invention relates to a process wherein recovery of insoluble recombinant polypeptides from bacterial periplasm is increased.
2. Description of Related Disclosures
Escherichia coli has been widely used for the production of heterologous proteins in the laboratory and industry. E. coli does not generally excrete proteins to the extracellular medium apart from colicins and hemolysin (Pugsley and Schwartz, Microbiology, 32: 3-38 (1985)). Heterologous proteins expressed by E. coli may accumulate as soluble product or insoluble aggregates. See FIG. 1 herein. They may be found intracellularly in the cytoplasm or be secreted into the periplasm if preceded by a signal sequence. How one proceeds initially in the recovery of the products greatly depends upon how and where the product accumulates. Generally, to isolate the proteins, the cells may be subjected to treatments for periplasmic extraction or be disintegrated to release trapped products that are otherwise inaccessible.
The secretion of recombinant proteins to the periplasmic space has numerous advantages over expression in the cytoplasm. The periplasmic space contains only 7 out of the 25 known cellular proteases (Swamy and Goldberg, J. Bacteriol., 149: 1027-1033 (1982); French and Ward, J. Chem. Tech. and Biol., 54 (3): 301 (1992)) and comprises only 4-8% of the total cell protein (Beacham, Int. J. Biochem., 10: 877-883 (1979)). The mature secreted protein does not include N-formyl methionine and the oxidative environment of the periplasm facilitates correct disulfide bonding and protein folding (Fahey et al., J. Mol. Evol., 10: 155-160 (1977)). Numerous heterologous proteins have been secreted to the periplasmic space of E. coli. Some have involved use of fusion proteins (VIIIa-Komaroff et al., Proc. Natl. Acad. Sci. USA, 75: 3727-3731 (1978); EP 6,694; and U.S. Pat. No. 4,336,336). Specific products prepared include antibody fragments (Pluckthun, Nature, 347: 497-498 (1990); WO 93/06217), ribonuclease A (Tarragona-Fiol et al., Gene, 118: 239-245 (1992)), HIV-1 receptor (Rochenbach et al., Appl. Microbiol. Biotechnol., 35: 32-37 (1991)), trypsin (Vasquez et al., J. Cell. Biochem., 39: 265-276 (1989)); human stefin A (Strauss et al., Biol. Chem. Hoppe Syeler, 369: 1019-1030 (1988)), xylanase (Bon-Joon et al., J. Microb. and Tech., 6: 414-419 (1996)), rat GM-CSF (Holowachuk and Ruhoff, Protein Exp. and Purification, 6: 588-596 (1995)), and interleukin-2 (Halfmann et al., J. Gen. Microbiol., 139: 2465-2473 (1993)).
The conventional isolation of heterologous polypeptide from gram-negative bacteria poses problems owing to the tough, rigid cell walls that surround these cells. The bacterial cell wall maintains the shape of the cell and protects the cytoplasm from osmotic pressures that may cause cell lysis; it performs these functions as a result of a highly cross-linked peptidoglycan (also known as murein) backbone that gives the wall its characteristic rigidity. A recent model described the space between the cytoplasmic and outer membranes as a continuous phase filled with an inner periplasmic polysaccharide gel that extends into an outer highly cross-linked peptidoglycan gel (Hobot et al., J. Bact., 160: 143 (1984)). This peptidoglycan sacculus constitutes a barrier to the recovery of any heterologous polypeptide not excreted by the bacterium into the medium.
To release recombinant proteins from the E. coli periplasm, treatments involving chemicals such as chloroform (Ames et al., J. Bacteriol., 160: 1181-1183 (1984)), guanidine-HCl, and Triton X-100 (Naglak and Wang, Enzyme Microb. Technol., 12: 603-611 (1990)) have been used. However, these chemicals are not inert and may have detrimental effects on many recombinant protein products or subsequent purification procedures. Glycine treatment of E. coli cells, causing permeabilization of the outer membrane, has also been reported to release the periplasmic contents (Ariga et al., J. Ferm. Bioenq., 68: 243-246 (1989)). These small-scale periplasmic release methods have been designed for specific systems. They do not translate easily and efficiently and are generally unsuitable as large-scale methods.
The most widely used methods of periplasmic release of recombinant protein are osmotic shock (Nosal and Heppel, J. Biol. Chem., 241: 3055-3062 (1966); Neu and Heppel, J. Biol. Chem., 240: 3685-3692 (1965)), hen eggwhite(HEW)-lysozyme/ethylenediamine tetraacetic acid (EDTA) treatment (Neu and Heppel, J. Biol. Chem., 239: 3893-3900 (1964); Witholt et al., Biochim. Biophys. Acta, 443: 534-544 (1976); Pierce et al., ICheme Research Event, 2: 995-997 (1995)), and combined HEW-lysozyme/osmotic shock treatment (French et al., Enzyme and Microb. Tech., 19: 332-338 (1996)). Typically, these procedures include an initial disruption in osmotically-stabilizing medium followed by selective release in non-stabilizing medium. The composition of these media (pH, protective agent) and the disruption methods used (chloroform, HEW-lysozyme, EDTA, sonication) vary among specific procedures reported. A variation on the HEW-lysozyme/EDTA treatment using a dipolar ionic detergent in place of EDTA is discussed by Stabel et al., Veterinary Microbiol., 38: 307-314 (1994). For a general review of use of intracellular lytic enzyme systems to disrupt E. coli, see Dabora and Cooney in Advances in Biochemical Engineering/Biotechnology, Vol. 43, A. Fiechter, ed. (Springer-Verlag: Berlin, 1990), pp. 11-30.
HEW-lysozyme acts biochemically to hydrolyze the peptidoglycan backbone of the cell wall. The method was first developed by Zinder and Arndt, Proc. Natl. Acad. Sci. USA, 42: 586-590 (1956), who treated E. coli with egg albumin (which contains HEW-lysozyme) to produce rounded cellular spheres later known as spheroplasts. These structures retained some cell-wall components but had large surface areas in which the cytoplasmic membrane was exposed.
U.S. Pat. No. 5,169,772 discloses a method for purifying heparinase from bacteria comprising disrupting the envelope of the bacteria in an osmotically-stabilized medium, e.g., 20% sucrose solution using, e.g., EDTA, lysozyme, or an organic compound, releasing the non-heparinase-like proteins from the periplasmic space of the disrupted bacteria by exposing the bacteria to a low-ionic-strength buffer, and releasing the heparinase-like proteins by exposing the low-ionic-strength-washed bacteria to a buffered salt solution.
There are several disadvantages to the use of the HEW-lysozyme addition for isolating periplasmic proteins. The cells must be treated with EDTA, detergent, or high pH, all of which aid in weakening the cells. Also, the method is not suitable for lysis of large amounts of cells because the lysozyme addition is inefficient and there is difficulty in dispersing the enzyme throughout a large pellet of cells.
Many different modifications of these methods have been used on a wide range of expression systems with varying degrees of success (Joseph-Liazun et al., Gene, 86: 291-295 (1990); Carter et al., Bio/Technology, 10: 163-167 (1992)). Although these methods have worked on a laboratory scale, they involve too many steps for an efficient large-scale recovery process.
Efforts to induce recombinant cell culture to produce lysozyme have been reported. EP 155,189 discloses a means for inducing a recombinant cell culture to produce lysozymes, which would ordinarily be expected to kill such host cells by means of destroying or lysing the cell wall structure. Russian Pat. Nos. 2043415, 2071503, and 2071501 disclose plasmids and corresponding strains for producing recombinant proteins and purifying water-insoluble protein agglomerates involving the lysozyme gene. Specifically, the use of an operon consisting of the lysozyme gene and a gene that codes for recombinant protein enables concurrent synthesis of the recombinant protein and a lysozyme that breaks the polysaccharide membrane of E. coli. 
U.S. Pat. No. 4,595,658 discloses a method for facilitating externalization of proteins transported to the periplasmic space of E. coli. This method allows selective isolation of proteins that locate in the periplasm without the need for lysozyme treatment, mechanical grinding, or osmotic shock treatment of cells. U.S. Pat. No. 4,637,980 discloses producing a bacterial product by transforming a temperature-sensitive lysogen with a DNA molecule that codes, directly or indirectly, for the product, culturing the transformant under permissive conditions to express the gene product intracellularly, and externalizing the product by raising the temperature to induce phage-encoded functions. JP 61-257931 published Nov. 15, 1986 discloses a method for recovering IL-2 using HEW-lysozyme. Asami et al., J. Ferment. and Bioeng., 83: 511-516 (1997) discloses synchronized disruption of E. coli cells by T4 phage infection, and Tanji et al., J. Ferment. and Bioeng., 85: 74-78 (1998) discloses controlled expression of lysis genes encoded in T4 phage for the gentle disruption of E. coli cells.
The development of an enzymatic release method to recover recombinant periplasmic proteins suitable for large-scale use is reported by French et al., Enzyme and Microbial Technology, 19: 332-338 (1996). This method involves resuspension of the cells in a fractionation buffer followed by recovery of the periplasmic fraction, where osmotic shock immediately follows lysozyme treatment. The effects of overexpression of the recombinant protein, S. thermoviolaceus α-amylase, and the growth phase of the host organism on the recovery are also discussed.
Further, E. coli mutants that leak various periplasmic enzymes have been isolated. For example, Lopes et al., J. Bacteriol., 109(2): 520-525 (1972) treated E. coli cells with a mutagen such as nitrosoguanidine, and mutants excreting periplasmic enzymes were selected by enzyme assay systems. Such mutants included those leaking ribonuclease I, endonuclease I, and alkaline phosphatase. It is believed that these mutants are deficient in some component of the outer bacterial membrane leading to an increase in the cells' permeability. In addition, several excreted periplasmic proteins have been separated from the culture medium by antibody precipitation or SDS-polyacrylamide gel electrophoresis in order to characterize these “periplasmic leaky” mutants. See, for example, Anderson et al., J. Bacteriol. 140(2): 351-358 (1979) and Lazzaroni and Portalier, J. Bacteriol. 145 (3): 1351-1358 (1981).
In a 10-kiloliter-scale process for recovery of IGF-I polypeptide (Hart et al., Bio/Technology, 12: 1113 (1994)), the authors attempted the typical isolation procedure involving a mechanical cell breakage step followed by a centrifugation step to recover the solids. The results were disappointing in that almost 40% of the total product was lost to the supernatant after three passes through the Gaulin homogenizer. Hart et al., Bio/Technoloqy 12: 1113 (1994). See FIG. 2 herein. Product recovery was not significantly improved even when the classical techniques of EDTA and HEW-lysozyme additions were employed.
While HEW-lysozyme is the only practical commercial lysozyme for large-scale processes, lysozyme is expressed by bacteriophages upon infection of host cells. Lysis of E. coli, a natural host for bacteriophages, for example the T4 phages, requires the action of two gene products: e and t. Gene e encodes a lysozyme (called T4-lysozyme for the T4 phage) that has been identified as a muramidase (Tsugita and Inouye, J. Biol. Chem., 243: 391 (1968)), while gene t seems to be required for lysis, but does not appear to have lysozyme activity. Gene t is required for the cessation of cellular metabolism that occurs during lysis (Mukai et al., Vir., 33: 398 (1967)) and is believed to degrade or alter the cytoplasmic membrane, thus allowing gene product e to reach the periplasm and gain access to the cell wall (Josslin, Vir., 40: 719 (1970)). Phage are formed by gene t-mutants, but lysis of the E. coli host does not occur except by addition of chloroform (Josslin, supra). Wild-type T4-lysozyme activity is first detected about eight minutes after T4 infection at 37° C., and it increases through the rest of the infection, even if lysis inhibition is induced. In the absence of secondary adsorption, cells infected by gene e mutants shut down progeny production and metabolism at the normal time, but do not lyse (Molecular Genetics of Bacteriophage T4, J. D. Karam, ed. in chief (American Society for Microbiology, Washington D.C., ASM Press, 1994), p. 398).
Recovery of insoluble IGF-I using T4-lysozyme was disclosed on Oct. 28, 1997 at the “Separation Technology VII meeting entitled ‘Separations for Clean Production’” in Davos, Switzerland, sponsored by the Engineering Foundation.
For controlling cost of goods and minimizing process time, there is a continuing need for increasing the total recovery of insoluble heterologous polypeptides contained in refractile particles from the periplasmic space of prokaryotes. Further, there is a need for culturing of E. coli cells to high cell densities as an important factor for achieving efficient recombinant heterologous polypeptide production.